Agronomy and Crop Science

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158–EF421160 and EU124518–EU124520) and rp gene sequences (EU277012–EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows (‘Ca. phytoplasma asteris’) subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of ‘Ca. phytoplasma asteris’-related strains associated with lily plants in Mexico. References: (1) R. F. Davis et al. Microbiol. Res. 158:229, 2003. (2) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (3) B. Duduk et al. Bull. Insectol. 60 2:341, 2007. (4) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004.

Citrus huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The disease is associated with three different species of fastidious α-proteobacteria, namely ‘Candidatus Liberibacter asiaticus’, Ca. L. americanus, and Ca. L. africanus (1). ‘Ca. L. asiaticus’ was first detected in South Florida in 2005 and has spread throughout the citrus-growing areas of Florida. ‘Ca. L. asiaticus’ is transmitted naturally by the Asian citrus psyllid, Diaphorina citri, and can also be transmitted by graft propagation and via various species of dodder (Cuscuta). HLB affects most if not all citrus and citrus relatives within the family Rutaceae (2), including the ornamental shrub Murraya paniculata (4). In addition, ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ can infect tobacco (Nicotiana xanthi) and periwinkle (Catharanthus roseus) (1,4). Here we report that ‘Ca. L. asiaticus’ can infect tomato (Lycopersicon esculentum) cvs. Manapal and FL47. Manapal and FL 47 plants grown from seed were placed adjacent to ‘Ca. L. asiaticus’-infected sweet orange (Citrus sinensis) plants with dodder (Cuscuta pentagona) already well established on them. Young dodder shoots still connected to the citrus were draped over the tomato plants and subsequently also became attached to the tomato stems. After 1 month, the tomato plants were detached from the citrus and most of the dodder removed. One month later, these tomato plants started to show vein clearing and subsequently the mature leaves became thicker and leathery. Some leaves showed blotchy mottle symptoms and some fruits became lopsided in a manner similar to HLB symptom expression on citrus. PCR amplification of the ‘Ca. L. asiaticus’ 16S rDNA with primers OI1/OI2c and the β-operon with primers A2/J5 (1) revealed the presence of ‘Ca. L. asiaticus’ DNA. Sequence analysis confirmed that the sequences of the cloned amplicons were identical to those from the HLB-infected citrus source plant. Both conventional and quantitative real-time PCR (3) revealed a much lower abundance of ‘Ca. L. asiaticus’ DNA in tomato as compared with ‘Ca. L. asiaticus’-infected citrus or periwinkle, indicating that ‘Ca. L. asiaticus’ bacteria multiplied at a lower titer in these tomato cultivars. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) S. E. Halbert et al. Fla. Entomol. 87:330, 2004. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) L. Z. Zhou et al. Plant Dis. 91:227, 2007.

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of ‘Ca. L. asiaticus’ in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of ‘Ca. L. asiaticus’ in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5′-AAGAGTTTGATCCTGGCTCAGGATT-3′/5′-CGTCCTTCATCGGCTCTT-3′), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5′-GAAACGACTGCTAAGACTGG-3′/5′-TGACGGGCGGTGTGTACAAACCCCG-3′) yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members (‘Candidatus Phytoplasma trifolii’) and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the ‘Ca. Phytoplasma trifolii’ reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a ‘Ca. Phytoplasma trifolii’-related strain. References: (1) M. Klein. Plant Dis. Rep. 54:735, 1970. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) M. Salehi et al. Plant Dis. 91:625, 2007. (4) K. Wang and C. Hiruki, Phytopathology 91:546, 2001.

Atalantia buxifolia (Poir.) Oliv., synonym Severinia buxifolia (Poir.) Ten. as commonly found in literature, is a common landscape plant and a popular Chinese medicinal herb known as Jiubingle or Dongfengjie. It remains unclear if this rutaceous plant could host ‘Candidatus Liberibacter asiaticus’, the pathogen of citrus Huanglongbing (HLB) in Guangdong, P. R. China. This information is important for HLB control in citrus because infected A. buxifolia could serve as a source of inoculum. In August of 1994, three A. buxifolia plants adjacent to a citrus experimental orchard of the South China Agricultural University at Guangzhou were found showing leaf mottle/yellowing symptoms. Two buds from each plant were grafted onto three mandarin trees (Citrus reticulata cv. Pongan) in a psyllid-proof screenhouse for indexing. By October of 1995, typical leaf mottle symptoms were observed in all three grafted trees compared with a healthy control. In March of 1996, one of the A. buxifolia plants was transferred to a screenhouse and has been maintained there. The leaf mottle/yellowing symptoms persisted but did not significantly affect plant growth. DNA was extracted from leaf samples in October 2006 by using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR using the general bacterial 16S rDNA primer set fDl/rD1 as the first round of amplification and primer set OI1/OI2c as second round amplification (1,3). After agarose gel electrophoresis and staining with ethidium bromide, an approximate 1.1-kb DNA band was detected in symptomatic samples but not healthy leaf samples of A. buxifolia and C. reticulata. XbaI digestion of the amplicons yielded approximate 500- and 600-bp fragments, characteristic of ‘Ca. L. asiaticus’. Similarly, a standard PCR with primer set A5/J2 (3) yielded an approximate 700-bp DNA band characteristic of ‘Ca. L. asiaticus’ from symptomatic samples only. To our knowledge, this is the first report of graft transmission and PCR detection of ‘Ca. L. asiaticus’ from A. buxifolia in Guangdong, P. R. China. This work also confirms the findings from Taiwan (2) that A. buxifolia could serve as a source of ‘Ca. L. asiaticus’. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) T.-H. Hung et al. Eur. J. Plant Pathol. 107:183, 2001. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.